Methods for using mutant RNA polymerases with reduced discrimination between non-canonical and canonical nucleoside triphosphates

Abstract

A method for synthesizing a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate using a mutant polymerase having a reduced discrimination between canonical and non-canonical substrates is disclosed. The method comprises incubating a template nucleic acid in a reaction mixture comprising the mutant nucleic acid polymerase and the appropriate canonical and non-canonical nucleoside triphosphates which are desired substrates for the mutant nucleic acid polymerase. The present invention is also a method of determining the sequence of a nucleic acid molecule using the mutant polymerase to create a nucleic acid molecule comprising at least one non-canonical nucleoside triphosphate.

We claim:  
     
       1. A method for determining sequence of a nucleic acid molecule, comprising the steps of: 
       a) synthesizing a nucleic acid molecule from a RNAP promoter sequence in a reaction mixture containing a mutant T7-type RNA polymerase in each of four separate reactions, wherein the T7-type RNA polymerase is selected from the group consisting of T3, φI, φIIH, W31, gh1, Y and A1122, and has a reduced discrimination between canonical and non-canonical nucleoside triphosphates, each reaction comprising at least four nucleoside triphosphates, wherein at least one nucleoside triphosphate has a nucleic acid base which is complementary to each of adenine, cytidine, guanine and uracil or thymine and a sugar with either a hydroxy or a hydrogen or a fluorine at the 2′-position, and further comprising a ddNTP, such that each of the four separate reactions forms a plurality of reaction products of differing length, the length of said reaction products indicating the positions or the type of base corresponding to the incorporated ddNTP, and  
       b) evaluating the reaction products so that the sequence of the template molecule may be deduced.  
     
     
       2. The method of  claim 1 , wherein each reaction comprises at least four nucleoside triphosphates chosen from the group consisting of ATP, CTP, GTP, and UTP or rTTP. 
     
     
       3. The method of  claim 1 , wherein each reaction comprises at least four nucleoside triphosphates chosen from the group consisting of dATP, dCTP, dGTP, dUTP, dTTP, 7-deaza-dGTP, dITP, 5-methyl-dCTP, and 5-hydroxy-methyl-dCTP. 
     
     
       4. The method of  claim 1 , wherein each reaction comprises at least four nucleoside triphosphates chosen from the group consisting of 2′-F-ATP, 2′-F-CTP, 2′-F-GTP, 2′-F-UTP, 2′-F-TTP, 2′-deaza-2′-F-GTP, 2′-F-ITP, 5-methyl-2′-F-CTP, and 5-hydroxymethyl-2′-F-CTP. 
     
     
       5. A method for determining the sequence of a nucleic acid molecule, comprising the steps of: 
       a) synthesizing a nucleic acid molecule by extending a primer, wherein at least part of the primer is complementary to a template molecule so as to anneal therewith, in a reaction mixture containing a mutant T7-type RNA polymerase in each of four separate reactions, wherein said mutant T7-type RNA polymerase has a reduced discrimination between canonical and non-canonical nucleoside triphosphates, each comprising at least four nucleoside triphosphates, wherein at least one nucleoside triphosphate has a nucleic acid base which is complementary to each of adenine, cytidine, guanine and uracil or thymine and a sugar with either a hydroxy or a hydrogen or a fluorine at the 2′-position, and further comprising a ddNTP, such that each of the four separate reactions forms a plurality of reaction products of differing length, the length of said reaction products indicating the positions of the type of base corresponding to the incorporated ddNTP; and  
       b) evaluating the reaction products so that the sequence of the template molecule may be deduced.  
     
     
       6. The method of  claim 5 , wherein the T7-type RNA polymerase is selected from the group consisting of T3, φI, φIIH, W31, gh1, Y and A1122. 
     
     
       7. The method of  claim 5 , wherein each reaction comprises at least four nucleoside triphosphates chosen from the group consisting of ATP, CTP, GTP, and UTP or rTTP. 
     
     
       8. The method of  claim 5 , wherein each reaction comprises at least four nucleoside triphosphates chosen from the group consisting of dATP, dCTP, dGTP, dUTP, dTTP, 7-deaza-dGTP, dITP, 5-methyl-dCTP, and 5-hydroxymethyl-dCTP. 
     
     
       9. The method of  claim 5 , wherein each reaction comprises at least four nucleoside triphosphates chosen from the group consisting of 2′-F-ATP, 2′-F-CTP, 2′-F-GTP, 2′-F-UTP, 2′-F-TTP, 2′-deaza-2′-F-GTP, 2′-F-ITP, 5-methyl-2′-F-CTP, and 5-hydroxymethyl-2′-F-CTP. 
     
     
       10. The method of  claim 1 , wherein a dinucleotide or trinucleotide for initiation of de novo acid synthesis is added to the reaction mixture. 
     
     
       11. The method of  claim 1 , wherein at least one of the nucleoside triphosphates in the reaction mixture is modified to contain a radioactive or non-radioactive label. 
     
     
       12. The method of  claim 1 , wherein the ddNTP in the reaction mixture is modified to contain a radioactive or non-radioactive label. 
     
     
       13. The method of the  claim 10 , wherein the dinucleotide or trinucleotide in the reaction mixture is modified to contain a radioactive or non-radioactive label. 
     
     
       14. A method for determining sequence of a nucleic acid molecule, comprising the steps of: 
       a) synthesizing a nucleic acid molecule from a RNAP promoter sequence in a reaction mixture containing a mutant T7-type RNA polymerase, wherein the T7-type RNA polymerase is selected from the group consisting of T3, φI, φIIH, W31, gh1, Y and A1122, in each of four separate reactions, wherein said mutant T7-type RNA polymerase has a reduced, discrimination between canonical and non-canonical nucleoside triphosphates, each reaction comprising at least four nucleoside triphosphates, wherein at least one nucleoside triphosphate has a nucleic acid base which is complementary to each of adenine, cytidine, guanine and uracil or thymine and a sugar with either a hydrogen or a fluorine at the 3′-position, and further comprising a rNTP;  
       b) treating the nucleic acid products of the reactions so as to bring about hydrolysis of the rNTP has been incorporated, whereby a plurality of reaction products of differing length are formed, the length of said reaction products indicating the positions of the type of base corresponding to the incorporated rNTP; and  
       b) evaluating the reaction products so that the sequence of the template molecule may be deduced.  
     
     
       15. The method of  claim 14 , wherein the nucleic acid synthesis is part of or coupled to a method for nucleic acid amplification. 
     
     
       16. A method for determining the sequence of a nucleic molecule, comprising the steps of: 
       a) synthesizing a nucleic acid molecule by extending a primer, wherein at least part of the primer is complementary to a template molecule so as to anneal therewith, in a reaction mixture containing a mutant T7-type RNA polymerase in each of four separate reactions, wherein said mutant T7-type RNA polymerase has a reduced discrimination between canonical and non-canonical nucleoside triphosphates, each reaction comprising at least four nucleoside triphosphates, wherein at least one nucleoside triphosphate has a nucleic acid base which is complementary to each of adenine, cytidine, guanine and uracil or thymine and a sugar with either a hydrogen or a fluorine at the 2′-position, and further comprising a rNTP;  
       b) treating the nucleic acid products of the reactions so as to bring about hydrolysis of the phosphodiester backbone at all sites where a rNTP has been incorporated, whereby a plurality of reaction products of differing length are formed, the length of said reaction products indicating the positions of the type of base corresponding to the incorporated rNTP; and  
       c) evaluating the reaction products using any of the methods common in the art for separating and detecting products of sequencing reactions so that the sequence of the template molecule may be deduced.  
     
     
       17. The method of  claim 16 , wherein the T7-type RNA polymerase is selected from the group consisting of T3, φI, φIIH, W31, gh1, Y and A1122. 
     
     
       18. The method of  claim 16 , wherein the nucleic acid synthesis is part of or coupled to a method for nucleic acid amplification.